Our objective is to use simian virus 40 (SV40) and polyoma virus (PV) chromosomes as a model system to learn, at the molecular level, how mammalian cells replicate their chromosomes, and how chromosome replication is related to cell proliferation. We will determine where and how DNA sequences affect the mechanism of initiation of DNA replication by determining the precise locations and structures of 5 feet ends of nascent DNA chains in and around the ori region of SV40 and PV DNA replication, comparing wild type with various mutants in this region. Are all DNA chains initiated by RNA primers, or is there a unique event such as a "cap" or nucleotide linked protein? We will study the biochemistry of initiation of DNA synthesis at ori and elsewhere (i.e., Okazaki fragments at replication forks) by reconstituting the events involved in initiation, elongation and ligation of Oazaki fragments from purified proteins and nucleic acids, by attempting to initiate Okazaki fragment synthesis in duplex DNA containing the ori region, and by determining under what conditions subcellular systems from virus infected cells will continue to initiate viral DNA replication. We will investigate the role of T antigen in initiation of DNA synthesis by mapping its location(s) on native replicating SV40 and PV chromosomes. We will relate chromatin structure to DNA replication and transcription by developing and exploiting a new approach to mapping the locations of nucleosomes, quantitatively, on a unique DNA sequence using SV40 and PV chromosomes. We will investigate the relationship between specific DNA sites that arrest replication forks, sites that arrest the progress of DNA polymerase alpha, sites that promote nucleosome binding, and the problems of termination of DNA replication and the movement of replication forks. Taken together with data gathered previously by this and other laboratories, we hope to provide a comprehensive view of the sequence of molecular and enzymological events in the replication of these miniature mammalian chromosomes.